Mild shading promotes sesquiterpenoid synthesis and accumulation in Atractylodes lancea by regulating photosynthesis and phytohormones

Atractylodes lancea rhizome (AR) has high medicinal and economic value. A previous study has reported that the accumulation of sesquiterpenoids in AR has obvious advantages under bamboo canopy. A concrete shade value to promote the cultivation of high-quality AR has not been established. In this study, 80% shading was screened at six different light intensities (100%, 80%, 60%, 40%, 20%, 7%), and the mechanism was explored in terms of photosynthetic efficiency and phytohormones levels. The results indicated that the total sesquiterpenoid content of 80% mild shading increased by 58%, 52%, and 35%, respectively, compared to 100% strong light in seedling, expansion, and harvest stages and increased by 144%, 178%, and 94%, respectively, compared with 7% low light. The sesquiterpenoids hinesol and β-eudesmol contributed approximately 70% to the differential contribution ratio between mild shading and strong light (100%) or between mild shading and low light (7%). Furthermore, HMGR, DXR, and FPPS genes, which regulate sesquiterpenoid synthesis, were significantly upregulated in 80% mild shading. Transpiration rate; the intercellular CO2 concentration; net photosynthetic rate; and levels of jasmonic acid, abscisic acid, and gibberellin were strongly correlated (r > 0.85) with sesquiterpenoid accumulation. Cis-acting elements responding to light and phytohormones were present within the promoter regions of HMGR, DXR, and FPPS. Therefore, 80% shading promotes the synthesis and accumulation of sesquiterpenoids in AR by regulating photosynthetic efficiency and phytohormone production, thereby promoting transcriptional expression.


Results
Mild shading facilitates sesquiterpenoid accumulation and growth in Atractylodes lancea rhizome. To determine a concrete shading value for the production of high-quality and high-yielding AR, we examined the major compounds, including the sesquiterpenoids hinesol (Hin), β-eudesmol (Edu), and atractylone (Atl), and the polyacetylene atractylodin (Atd), as well as the biomass of AR at different growth stages ( Fig. 1A-C) under various light intensities. The sum of these four volatile oils as the total volatile oil content was subsequently analyzed. The results revealed that the accumulation of volatile oils was significantly different (p < 0.05) across seedling, expansion, and harvesting stages under different light intensities. The common features at the three life stages were that the total volatile oil content was the highest under 80% mild shading and the lowest under 7% low light intensity. At 80% mild shading, the total volatile oil content in seedling, expansion, and harvest stages increased by 58%, 52%, and 35%, respectively, compared to 100% strong light. Thus, excessive light inhibited the accumulation of volatile oils. Compared to 7% low light, the increased accumulation of volatile oils at 80% mild shading reached 144%, 178%, and 94% from seedling to harvesting stages. We found that the weaker the light intensity (20% and 7%), the lower the volatile oil accumulation. In addition, the biomass indicators of fresh and dry weights (Fig. 1G,H) indicated that the response to different light intensities yielded trends that were consistent with volatile oil accumulation. Thus, both quality and yield of AR can be achieved under 80% mild shading.
To further investigate the component that contributes the most to the total volatile oil accumulation due to changes in light intensity, individual compounds were analyzed at different life stages (Fig. 1D-F). The results revealed that each compound was significantly affected by changes in light intensity at different stages ( Fig. 1D-F). Statistically, the sum of the difference contribution ratio (DCR) of the three sesquiterpenoids Hin, Edu, and Atl was greater than 90% of the total variation at any stage of growth, with less than 10% of Atd. Of these, Hin and Edu contributed approximately 70% to the total variation (Table S-1). Therefore, the effect of different light intensities on the accumulation of Hin and Edu was higher than that of Atl and Atd. Sesquiterpenoids, especially Hin and Edu, could reliably determine the variations in the total volatile oil content. Compared to 100% strong light, Hin increased by 60%, 52%, and 30% and Edu increased by 40%, 60%, and 45% at 80% mild shading, respectively, from seedling to harvesting stages. Compared to 7% low light, Hin increased by 111%, 185%, and 92%, and Edu increased by 182%, 224%, and 81% at 80% mild shading, respectively, from seedling to harvesting stages. In addition, we also measured the levels of the key enzyme genes of sesquiterpenoid biosynthesis during the expansion stage of AR. The expression levels of HMGR, DXR, and FPPS in the mevalonic acid (MVA) pathway and methylerythritol phosphate (MEP) pathway were measured by qRT-PCR. The results revealed that the expression of these genes under different light intensities significantly differed among the groups (Fig. 1I). The expression of each gene was the highest under 80% mild shading, that is, greater than 100% strong light, and the lowest under 7% low light. The differential expression patterns of these genes under different light 80% mild shading enhances photosynthetic efficiency in A. lancea, whose trend was affected by changes in light intensity, consistent with sesquiterpenoid accumulation. The intercellular CO 2 concentration (C i ), transpiration rate (T r ), net photosynthetic rate (P n ), and stomatal conductance (G S ) as gas exchange parameters were significantly different at the different light intensities. The C i , T r , P n , and G s values of 80% mild shading were the highest (Fig. 2), increasing by 30%, 33%, 27%, and 27%, respectively, compared to 100% strong light and increasing by 45%, 369%, 347%, and 81%, respectively, compared to 7% low light. The results showed that between 100% strong light and 80% mild shading, the differences of the four photosynthetic parameters are close to 30%. While between 7% low light and 80% mild shading, the changes of T r , P n are far higher than C i, G s . As the degree of shade increased, light utilization decreased starting at 80% light intensity. The results showed that mild shading promotes photosynthetic efficiency in A. lancea, and the trend of variation is consistent with sesquiterpenoid accumulation, which is affected by changes in light intensity.  Effects of different light intensities on photosynthetic parameters, namely, the intercellular CO 2 concentration (C i ), transpiration rate (T r ), net photosynthetic rate (P n ), and stomatal conductance (G s ) in A. lancea. Lower-case letters represent significant differences (one-way ANOVA, p < 0.05). www.nature.com/scientificreports/ 80% mild shading significantly enhances the phytohormones ABA and GA 3 in A. lancea, whose trend was affected by changes in light intensity, consistent with sesquiterpenoid accumulation. Phytohormones play important roles in signal transduction and secondary metabolism in plants. We measured the levels of common phytohormones in root (R) and leaf (L), as shown in Fig. 3. Data analysis found that the levels of JA, salicylic acid (SA), ABA, and gibberellin (GA 3 ) under 80% mild shading were significantly (p > 0.05) higher than that of 100% and 7% light intensities, regardless of the plant part (Fig. 3). Furthermore, JA, SA, ABA, and GA 3 levels in 80% mild shading were 1.7, 2.9, 3.1, and 2.2-fold higher than those in 100% strong light, and 4.2, 3.2, 22.9, 19.8-fold higher than those in 7% low light in root (Fig. 3A). And JA, SA, ABA, and GA 3 levels in 80% mild shading were 4.2, 3.2, 21.2, and 19.8-fold higher than those in 100% strong light, and 7.8, 2.9, 11.8, 2.2-fold higher than those in 7% low light in leaves (Fig. 3B). The results showed that between 100% strong light and 80% mild shading, the phytohormone change folds of ABA and GA 3 are higher than other phytohormones in leaves. While between 7% low light and mild shading, in root the phytohormone change folds of ABA and GA 3 are higher than other phytohormones. Therefore, under strong light stress, ABA and GA 3 in leaves were more responsive, while under weak light stress, ABA and GA 3 in roots were more responsive. Other phytohormones content also fluctuated with different light intensity changes, but the fluctuation range was relatively small. In addition, all phytohormone levels gradually decreased with weakening light intensity starting at 80% light intensity (Fig. 3). The results indicated that the changes in the phytohormone levels are consistent with those of sesquiterpenoid accumulation. In addition, we compared the phytohormone response levels in root and leaf. When the phytohormone change fold (HCF) of roots to leaves was equal to 1, the hormone response level was similar in both root and leaf. When the HCF was higher than 1, the hormone response level was higher in root than in leaf, indicating that hormonal stress in roots is more sensitive to changes in light intensity. By contrast, when the HCF was lower than 1, the hormone response level was lower in root than in leaf, indicating that hormonal stress in leaves is more sensitive to changes in light intensity. Table 1 shows that JA was more sensitive to light intensity changes in leaves than in roots, while ABA was higher responseive to light intensity changes in roots than in leaves.   www.nature.com/scientificreports/ sities. As shown in Table S-2, most indexes were significantly correlated with the volatile oil content (p < 0.01). The correlation coefficient represents the strength of the correlation between the two indexes. Correlation coefficients greater than 0.85 were used as the screening criteria. As shown in Fig. 4, the values of C i , T r and P n were strongly correlated with the sesquiterpenoids hinesol and β-eudesmol (r > 0.85). From the correlation network, we found that R-ABA, R-GA 3 and L-JA showed the highest correlation with the accumulation of sesquiterpenoids. Based on the results of photosynthetic efficiency test, phytohormone test and Pearson correlation analysis, Tr and Pn in photosynthesis, GA3 and ABA in root, and JA in leaf that are not only significantly responsive to light intensity changes but also significantly related to sesquiterpenes are screened out.

Ci, Tr, and Pn in photosynthesis
Promoter regions of HMGR, DXRs, and FPPS contain both light and phytohormone cis-acting regulatory elements. HMGR, DXR, and FPPS have been reported as key enzyme genes involved in biosynthesis of sesquiterpenoids. Therefore, we analyzed the cis-acting elements within the upstream promoter regions (2000 bp) of the three genes which had been selected for qRT-PCR analysis. The relevant sequences are shown in Fig. S-1. The results indicated that HMGR, DXR, and FPPS promoter regions included several light and phytohormone regulatory elements ( Table 2), confirming that sesquiterpenoid genes may co-regulated by light and phytohormones.

Discussion
Mild shading facilitates the accumulation of volatile oils and growth in A. lancea. Synthesize multiple results showed that Tr and Pn may be the key extrinsic factors affecting the synthesis and the accumulation of the sesquiterpenoids hinesol and β-eudesmol. We know that photosynthesis is an important indicator of growth and development as well as stress resistance in plants 36,37 . This study showed that Pn, which reflects plant organic matter accumulation, was the highest under 80% mild shading. In addition, AR also accumulates more biomass under this condition. The substances produced by photosynthesis far exceeded the substances consumed by the plant for its own life activities, indicating that it grew well. The 100% strong light provided more light energy than 80% mild shading, but the biomass of 100% strong light was lower than that under 80% mild shading. This can be explained by the fact that high light and high temperature in the summer cause severe leaf burns, and photosynthetic structures are irreversibly damaged 14 , leading to an imbalance of Tr and a decline of Pn. According to Farquhar and Sharkey, who commented on the reduced photosynthetic rate, when Pn and Gs decrease, if Ci also decreases, this indicates that the reduction of Gs is the main reason for the reduced photosynthetic rate 38 . Meanwhile, under the long-term regulation of shaded environments, stomatal density can decrease, leading to the decline of Gs 39 . Furthermore, Gs is the main factor of Tr intensity and Pn efficiency. Combined with the results of this study, under conditions of < 80% mild shading, with the increase of shading degree, photosynthetic efficiency was mainly affected by stomatal limitations. Studies have demonstrated that low light can reduce photosynthesis by inhibiting Ci and Gs to suppress secondary metabolic processes such as phycocyanin synthesis and yield in crops such as wheat 40,41 . Therefore, under 80% mild shading, A. lancea maintained the best photosynthesis state, and it was more capable of resisting the fluctuations of the external environment to produce more sesquiterpene secondary metabolites in a long-time environment. In addition to having better resistance www.nature.com/scientificreports/ to the environment at 80% mild shading, more organic substances accumulated in AR, thereby providing the elements needed for energy production such as acetyl coenzyme A, malonyl coenzyme A, and ATP 42 using for sesquiterpenoid biosynthesis.

Light-hormone interactions regulate key enzyme gene expression in sesquiterpenoid synthesis and further influence sesquiterpenoid accumulation. As physiological and biochemical indicators, phytohormones reflect the intrinsic expression of plant life activities. The Pearson correlation results
showed that the accumulation of sesquiterpenoids is strongly related to the changes of multiple hormones at different light intensities. Our findings are consistent with the results that the promoters of three key enzyme genes in the sesquiterpenoid synthesis pathway contain multiple cis-acting elements of phytohormones. Chuanchao and colleagues demonstrated that JA can up-regulate the expression of sesquiterpenoid genes through fungal induction; it also has functional interactions with SA in plant defense 43 . On the other hand, ABA and GA 3 up-regulate DXS and HMGR genes under bacterial induction and promote the accumulation of volatile compounds 44,45 . It was also found that blocking the production of just one hormone did not completely inhibit sesquiterpenoid production 43 , suggesting that sesquiterpenoid synthesis is not dependent on a single signaling event and that multiple phytohormones work together to regulate their synthesis. These findings reveal that multiple phytohormones showed good correlation with the accumulation of sesquiterpenoids. A previous molecular study demonstrated a relationship between light and several hormone signaling pathways via signaling integrators (PIFs, HY5) 46 . It has also been demonstrated that the function of JA in artemisinin is dependent on the presence of light 47 . Therefore, light may be the basic condition required to induce changes in phytohormone levels. However, A. lancea maintains different phytohormone levels, and their responses are the same at different light intensities. The levels were higher in mild shading, and lower in low light shading. This may be related to the growth inhibitory mechanism used by A. lancea under the stress of strong light or weak light for a long time to maintain a longer lifespan. In summary, the results of this study suggest that mild shade induces changes in the phytohormones ABA, and GA 3 , JA, which regulate the expression levels of key enzyme genes for sesquiterpenoid synthesis and ultimately influence the changes in bioactive components. Our findings revealed that ABA decreases under strong light, which is inconsistent with the results of a previous study that reported ABA unaffected by light signals 48 . Under long-term high light conditions, the leaves of A. lancea showed burning and yellowing as well as poor growth. Abscisic acid is a hormone that triggers leaf abscission, so ABA can be used as an indicator of the abscission of injured leaves, indicating that ABA levels change with plant growth and development. In addition, it was found that the content of ABA in roots was significantly higher than that in leaves. Currently, ABA is synthesized at several sites, such as roots and leaves, and translocated to its sites of action, which includes guard cells 49 . Given that plants have multiple ABA transporters, ABA transport is dynamically regulated under various growth conditions, which regulates root system expansion 50,51 .
In addition, we found that the promoters of key enzyme genes that regulate the synthesis of sesquiterpenoids contain numerous light response elements. Several studies have demonstrated that light can directly regulate the synthesis of isoprene, the precursor of sesquiterpenoids 52 . Therefore, in the future, studies should focus on the significance of light, which directly regulates the synthesis of sesquiterpenoids via the downstream factors of the light response.

Conclusion
In conclusion, 80% mild shading is a proper shade condition suitable for guiding stable and sustainable production of high-quality AR. It promotes the accumulation of total volatile oils mainly by regulating the biosynthesis of the sesquiterpenoids hinesol and β-eudesmol. Mild shading promotes the synthesis and accumulation of the medicinal compound in A. lancea via integration and regulation of photosynthesis and phytohormones. In addition, light and phytohormones are important internal and external factors affecting plant growth, development, and metabolism, although the direction of molecular research of A. lancea has been slow. Thus, this study provides a solid foundation upon which future studies can be designed. In the future, the relationship between photosynthesis (Tr, Ci, Pn), multiple hormones (ABA, GA 3 , JA), and sesquiterpenoids (hinesol, β-eudesmol) at the molecular level should be studied, laying the foundation for the regulation and the improvement of AR quality.

Materials and methods
All plant experiments were performed in accordance with relevant guidelines and regulations. Approximately 500 mg of each powdered sample was obtained and placed in a 50-mL centrifuge tube. After adding 25 mL of n-hexane (purity ≥ 95%, Beijing Chemical Works, Beijing, China), ultrasonic extraction was conducted at 40 kHz for 30 min. The samples were centrifuged at 3000 r min − 1 for 10 min, and the supernatants were collected. The pellet residues were suspended, and 25 mL of n-hexane was continuously added and extracted. This process was repeated, and after two rounds of centrifugation, the supernatants were combined, mixed well, and finally diluted with n-hexane to 50 mL. After filtration through an organic membrane, the samples were analyzed by gas chromatography-mass spectrometry (GC-MS) 53 . A TRACE 1310 GC instrument coupled to a TSQ 8000 mass spectrometer (Thermo Fisher Scientific) was used. An Agilent DB-5MS capillary column (0.25 mm × 30 m, 0.25 μm) was used; the carrier gas was helium gas, and the flow rate was 1 mL min − 1 . The injection mode was split flow (ratio 50:1), the injection port temperature was 240 ℃, and the injection volume was 1 μL. The temperature was increased to 120 °C for the first 2 min, and then to 240 °C at a rate of 5 °C min -1 , and then maintained at 240 °C for 5 min. The other parameters were as follows: EI (ionization voltage: 70 V, ion source 230 ℃, quadrupole temperature: 150 ℃) and MSD data acquisition mode (scanning ion range m/z 40-500). Figure 5 shows the GC-MS chromatograms of the four major volatile compounds in A. lancea 53 . www.nature.com/scientificreports/ Expression of genes encoding key enzymes involved in the biosynthesis of sesquiterpenoids in the expansion stage. The fresh rhizome samples of the expansion period, a mixture of roots, and leaves of A. lancea were stored in a small liquid nitrogen tank in the field and used for RNA extraction. Approximately 50 mg of leaves and roots were mixed and RNA was extracted with the TransZol UP Plus RNA Kit (TransGen Biotech Co., Beijing, China). The RNA was reverse-transcribed to obtain cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech Co.). Primers of genes encoding key enzymes involved in the sesquiterpenoid biosynthesis pathway (DXR, HMGR, and FPPS) were designed based on our transcriptome database (Table S-3). Real-time quantitative PCR (qRT-PCR) was conducted according to the instructions of the TransStart Top Green qPCR SuperMix Kit (TransGen Biotech Co.), and EF-1α served as the internal reference gene.
Measurement of the biomass of A. lancea at different developmental stages. Rhizome biomass, including the fresh weight (FW) and dry weight (DW) of A. lancea at seedling, expansion, and harvest stages was measured. The aerial parts and roots of the harvested A. lancea samples were removed, and the weight of the underground rhizomes was measured. There were ten replicates for each treatment group.
Determination of the photosynthetic parameters of A. lancea leaves at the expansion stage. The leaf photosynthetic parameters of A. lancea, including the intercellular CO 2 concentration (C i ), transpiration rate (T r ), net photosynthetic rate (P n ), and stomatal conductance (G s ), were determined under different light intensities using a Li-6400 portable photosynthesis instrument on a sunny day from 9:00 to 11:00 a.m. and 2:00 to 4:00 p.m. when plants were in the expansion stage. A. lancea leaves fully exposed to light and at the same positions were randomly selected. For each group, five plants were selected, and measurements were taken from two leaves on each plant. Each leaf was measured three times. lancea 53 , and each group had three replicates. After removing the surface dirt, the roots and leaves were immediately ground in a liquid nitrogen dry ice bath, weighed, and added to a 2-mL centrifuge tube. Next, 0.5 mL of isopropyl alcohol:water:HCl (2:1:2) extract was added to the centrifuge tube and mixed (4 ℃, 100 r min −1 ) for 30 min, and 1 mL of dichloromethane was then added to the centrifuge tube and mixed for 30 min. The samples were centrifuged (4 ℃, 13,000 r min −1 ) for 5 min, and 0.9 mL of the organic phase layer (middle layer) was absorbed and blow-dried with nitrogen. Next, 200 µL of a 50% methanol-water solution was added, followed by centrifugation (4 °C, 13,000 r min -1 ) for 15 min. The supernatant was then extracted for UPLC-MS/MS analysis. Chromatography was conducted with a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm). The flow phase was 0.05% formic acid water-acetonitrile (Thermo Fisher Scientific). The linear gradient elution was as follows: 0-0.3 min, 10% acetonitrile; 0.3-3 min, 10-60% acetonitrile; 3-6 min, 60-95% acetonitrile; and 6-6.2 min, 10% acetonitrile. The flow rate was 0.6 mL min -1 , and the column temperature was 4 ℃. The sample tray temperature was 4 ℃, the injection volume was 10 µL, and the analysis time was 7 min. The mass spectrometry conditions were as follows: the ionization mode was electrospray ion source (ESI), the scanning mode was negative ion scanning, the monitoring method was multi-response monitoring (MRM), the pressure of the air curtain was 30 psi, the ionization voltage was − 4500 V, the temperature of the ion source was 550 ℃, the spray gas pressure was 50 psi, and the auxiliary heating gas pressure was 50 psi. Data processing was conducted with Multi Quant Software.

Determination of the endogenous hormone content in roots and leaves of
Analysis of cis-acting elements in the predicted promoter regions of HMGR, DXR, FPPS genes that regulate sesquiterpenoid biosynthesis. Using the genomic data from this experimental platform, the upstream predicted promoter regions of HMGR, DXR, and FPPS was obtained by TBtools, and the potential cis-acting elements were predicted using Plantcare Software (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/).

Data analysis
Microsoft Excel 2016 and SPSS v26.0 Software (IBM, Armonk, NY, USA) were used for statistical and correlation analyses. The results were expressed as means ± standard deviations (SD). One-way analysis of variance (ANOVA), followed by the Fisher's protected least significance difference test, was performed to determine the main effects. The figures in the manuscript were created with GraphPad Prism Software and Adobe Illustrator CS6. The graphs for Pearson correlation network diagram were prepared with Cytoscape Software.

Data availability
The original contributions presented in this study are included in the article/supplementary material, and further inquiries can be directed to the corresponding authors.